PROJECT SUMMARY Microorganisms produce structurally diverse molecules, many of which have been successfully repurposed by mankind as pharmaceutical agents. These molecules are manufactured by multi-enzyme assemblies, which use acyl carrier proteins (ACPs) to modify and transfer chemical intermediates. Rational redesign of natural enzyme assemblies presents an exciting possible route to produce new antibiotics, but the success of any redesign approach depends on a thorough understanding of what leads to chemically productive hybrid ACP- enzyme interactions. The goal of this study is to understand how ACPs interact with both their molecular cargoes and partner enzymes. The dynamic structures of ACPs and the transient nature of their protein interactions make this class of proteins particularly challenging to study. Therefore, ACP interactions will be observed using site-specific vibrational spectroscopy and sedimentation velocity experiments, an innovative approach that is sensitive to fast conformational changes and weak protein-protein interactions. Information from this work will be analyzed in the context of data acquired from traditional methodologies to identify candidate hybrid ACP-ketosynthase partners capable of producing molecules of novel structure. The ability of the hybrid pairs to produce polyketides will be evaluated. Results from these studies will guide the future biosynthesis of novel small molecules with potential pharmaceutical activity.